One of the cellular actions of estrogen is to attenuate the mu-opioid response of hypothalamic neurosecretory neurons which control anterior pituitary secretions. Since estrogen appears to decrease the number of functional mu-opioid receptors in a manner similar to chronic morphine treatment, this proposal focuses on the common cellular components affected by these drugs and their interaction. Hypothalamic slices will be prepared from ovariectomized female guinea pigs which have been treated with estrogen or oil (control) and morphine or placebo. In the first series of experiments, we will study the interaction of chronic morphine and estrogen in altering the mu-opioid mediated membrane hyperpolarization of identified arcuate (ARC) neurons by measuring the shift in the mu-opioid dose response curve in the four groups of animals. We will also determine the timecourse of estrogen's actions by treating the slices in vitro with estrogen. Finally, we will measure the effects of chronic morphine on a heterologous receptor response (GABA/B) which is coupled to the inwardly rectifying K+ channel and affected by estrogen. In Experiments 2, we will study the effects of mu-opioid agonists on supraoptic (SON) vasopressin and oxytocin magnocellular neurons by ascertaining the specific K+ and Ca2+ conductances activated by mu-opioid agonists. We will characterize the effects of estrogen and chronic morphine on mu-opioid response in SON neurons. We will also identify and characterize the opioid synaptic input onto SON neurons using a hypothalamic explant preparation. In Experiment 3, we will study the cellular mechanism by which estrogen and morphine decrease the efficacy of the actions of mu-opioid agonists in ARC and SON neurons by measuring the expression of Galpha/i and Galpha/o mRNA and protein using in situ hybridization and immunocytochemistry. Lastly, electrophysiological experiments will be done in ARC and SON neurons using the whole-cell patch recording technique to study the role of G-proteins in the estrogen- mediated attenuation of the mu-opioid response. It is envisioned that this multidisciplinary approach will not only elucidate the physiological mechanisms by which estrogen alters opioidergic tone during the reproductive cycle of the mammal but the interaction of morphine and estrogen to down regulate opioid input in CNS neurons.